Team:Stanford/Notebook/Lab Work/Week 1

6/28 Monday

General Meeting Notes

• Wiki stuff
  • Everyone should sign up on 2010.igem.org website
    • Take advantage of the new calendar system
    • Need to transfer current website over
  • Current wiki needs a new vision
    • Francisco + Alex assembling wiki committee
• Transform the first wave of cells
  • Pick colonies
  • Colony PCR
  • Sequence parts to validate
    • Use the Smolke sequencing network
• Characterize promoters
  • Digest, ligate
  • Need to acquire enzymes
  • Need an PO account.
    • Email Jeanne
• Get competent cells
• Getting DNA
  • Assembly PCR
  • PCR from host cells
  • Synthesize de novo
• RNA team
  • Use RNAstructure
    • Maybe use ViennaRNA or dinamelt
  • Talk to Arkin and Berkeley Postdocs
  • Finalize sRNA library
  • 9kb plasmid
• Meeting scheudle
  • Monday morning is good for logistics
  • Group meetings for presentations
    • One person presents their work in the context of everything else
  • Journal club
  • Karina will set up a doodle

Alex's Notebook

• Transformations using DH5alpha cells from Graham (also have TOP10)

• Protocol:
  • 1) Take out competent E.coli cells from –80 C freezer.
  • 2) Turn on water bath to 42 C.
  • 3) Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar).
  • 4) KEEP EVERYTHING ON ICE UNLESS OTHERWISE STATED.
  • 5) Add 50 ng or 2.5/5 ul of circular DNA into E.coli cells. DO NOT PIPETTE UP OR DOWN; STIR INSTEAD! Incubate on ice for 15/30 min.
  • 6) Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
  • 7) Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
  • 8) Add 1 ml of SOC. Incubate tubes for 1/2 hour(s) at 37 C.
  • 9) Spread about 100/200 ul of the resulting culture on LB plates (with the appropriate antibiotic added – usually Ampicillin or Kanamycin). Grow overnight.
  • 10) Pick the colonies about 12-16 hours later.

Parts:  Plasmids: - 1A2  P1, 11P, BBa_J04450. 100-300, pMB1, 2079. - 1A3  P1, 1C, Ba_J04450. 100-300, pMB1, 2157. - 4A5  P1, 1G, BBa_J04450. ~5, pSC101, 3395. - 3C5  P1, 3C, BBa_J04450. 10-12, p15A, 2738. - 2K3  P1, 5C, Insert: BBa_J04450, star. Length: 4425 bp.

 Promoters: - Bad (BBa_I0500). Input(s): L-arabinose. Output(s): PoPS. Need with mutated strain. Tested range: 2e-4% up to 2e-1%. Can probably go lower. (http://mic.sgmjournals.org/cgi/reprint/147/12/3241).  P1, 14N, Plasmid: pSB2K3. Length: 1210 bp. - TetR and LuxR (BBa_F2620). Input(s): C10HSL. Output(s): PoPS. Tested range: ~1e-7 up to 1e-4. Can probably go to a higher concentration. (http://partsregistry.org/wiki/images/f/fe/EndyFig1-F2620DataSheet.pdf)  P2, 6E, Plasmid: pSB1A2, star. Length: 1061 bp.

 Reporter: - Endy’s GFP (BBa_E0240).  P1, 12M Plasmid: pSB1A2, star. Length: 876 bp.

 Miscellaneous Parts: - GFP Producer Controlled by 3OC6HSL Receiver Device (BBa_T9002). For use with C10HSL (homoserine lactone; http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=17248|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC)  P2, 9A, Plasmid: pSB1A3, star. Length: 1945 bp.

6/29 Tuesday

Laura's Notebook

• made LB agar (with Alex and Chris)
  • made 1 500 mL bottle, 3 250 mL bottles, then autoclaved

6/30 Wednesday

Laura's Notebook

• Re-try failed transformation (failed 2X for Alex and Chris)
• Protocol followed:

1. get competent cells from -80oC freezer
2. set H2O bath to 42oC (already done)
3. add 50 uL cells to a 1.5 mL tube (keep tubes on ice)
4. add 50 ng or 2.5 uL circular DNA to E. Coli (variation: used 5.0 uL I0500 this time)
5. on ice 30 min
6. 24oC H2O bath 20 sec.
7. on ice 15 min
8. add 1 mL SOC, 2 hours at 37oC (rotating)
9. plate100uL on appropriate plate (kanamycin this time), incubate 12-16 hours before picking colonies

• variations: 200 uL plated this time, also plated on kan/X-gal/IPTG plate (plasmid inducible copy # by X-gal)

7/1 Thursday

Laura's Notebook

• did 8 minipreps from Chris'/Alex's cultures
• used Qiagen spin kit, with the following variations:

1. skipped "recommended" wash step
2. eluted with H2O

• Concentrations from Nanodrop (taken by Chris B.)

• helped Karina, Francisco with the transformations of parts

Francisco's Notebook

• Learned how to transform using electroporation.

7/2 Friday

Recap Meeting Notes

• Things to think about:
  • name of the device (see if electronic analogue exists)
  • decouple the small RNA binding from the target gene ("RSID" tags)
  • leadership structure for the team

Francisco's Notebook

• Miniprepped Parts. Nanodrop results below: